Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of each series of experiments in this application, preceding the claims.
Protein kinase C (PKC) functions in processes relevant to carcinogenesis, tumor cell metastasis, and apoptosis. Safingol, an optical isomer (the L-threo enantiomer) of dihydrosphingosine, is a specific inhibitor of PKC and may represent a novel target for anti-cancer therapy. Preclinical animal studies show that safingol alone has minimal effects on tumor cell growth, but combination of this compound with conventional chemotherapy agents dramatically potentiates their anti-tumor effects. It has been suggested that many chemotherapeutic agents exert their anti-tumor effects by inducing apoptosis.
A large body of evidence indicates a fundamental role for the involvement of protein kinase C (PKC), family members of serine/threonine protein kinases, in processes relevant to neoplastic transformation, carcinogenesis, and tumor cell invasion of surrounding tissues (1-3). Consequently, PKC may represent a novel target for anti-cancer therapy. Safingol, the L-threo enantiomer of dihydrosphingosine, is a specific inhibitor of PKC (4). Preclinical animal studies show that safingol is non-toxic at doses that achieve serum levels sufficient to inhibit PKC enzyme activity (5). While safingol has negligible impact on tumor cell growth in vivo, the combination of safingol with conventional chemotherapeutic agents such as doxorubicin and cisplatin significantly potentiates the anti-tumor effects of these drugs (6).
Based on these observations, safingol, used in combination with doxorubicin, has become the first PKC specific inhibitor to enter clinical trials. The mechanism by which safingol potentiates the activity of chemotherapeutic agents is unclear, although inhibition of P-glycoprotein phosphorylation and reversal of the multidrug resistant (mdr) phenotype have been suggested (7,8). While this hypothesis can explain the synergism achieved with combinations of safingol and doxorubicin, it does not explain the synergism reported for combinations of safingol with drugs that are not believed to produce resistance by the mdr mechanism (e.g., cisplatin) (6), nor does it explain safingol-induced effects that occur in tumor cell lines that do not express the P-glycoprotein (8). Therefore, pathways other than P-glycoprotein inhibition are likely to be involved in the safingol-mediated enhancement of chemotherapy.
It has been suggested that the anti-tumor activity of many chemotherapeutic agents (e.g., cisplatin and etoposide) is a consequence of their induction of apoptosis (9). In this context it has been proposed that activation of PKC acts as an antagonist to apoptosis, whereas inhibition of PKC promotes apoptosis (10-12). Thus, safingol-nediated potentiation of chemotherapy might be attributed to its PKC inhibitory effect, subsequently leading to increased apoptosis after drug-induced damage.
The present studies sought to determine the extent to which whether safingol by itself, or in combination with a specific chemotherapeutic drug (e.g. mitomycin-C, MMC), would promote apoptosis in gastric cancer cells. Furthermore, applicants investigated whether the p53 status of these cells influences the development of apoptosis after treatment with safingol and MMC.
This invention provides a method for screening protein kinase C inhibitors capable of potentiating apoptosis in tumor cells comprising steps of (a) contacting an amount of a protein kinase C inhibitors with tumor cells effective to potentiate apoptosis of tumor cells; (b) contacting the potentiated tumor cells of step (a) with an antitumor therapeutic agent; (c) determining the apoptosis of tumur cells; and (d) comparing the apoptosis determined in step (c) with apoptosis of same tumor cells which are only treated with the antitumor therapeutic agent, an increase in apoptosis indicating that the protein kinase C inhibitor is capable of potentiating apoptosis in tumor cells. This invention also provide the above method, wherein step (a) is carried out in the presence of the antitumor therapeutic agent.
This invention also provides the protein kinase C inhibitor capable of potentiating apoptosis in tumor cells as determined by the above-described methods.
This invention further provides a pharmaceutical composition comprising the protein kinase C inhibitor capable of potentiating apoptosis in tumor cells as determined by the above-described methods and a pharmaceutically acceptable carrier.
This invention provides a method for screening antitumor therapeutic agents suitable for combination therapy with a protein kinase C inhibitors capable of potentiating apoptosis in tumor cells comprising steps of: (a) contacting an amount of a protein kinase C inhibitor with tumor cells effective to potentiate apoptosis of tumor cells; (b) contacting the potentiated tumor cells of step (a) with an antitumor therapeutic agent; (c) determining the apoptosis of tumor cells; and (d) comparing the apoptosis determined in step (c) with apoptosis of same tumor cells which are only treated with the protein kinase C inhibitor, an increase in apoptosis indicating that the antitumor therapeutic agent is suitable for combination therapy with a protein kinase C inhibitor capable of potentiating apoptosis in tumor cells. In an embodiment, step (a) is carried out in the presence of the protein kinase C inhibitor. In another embodiment, the antitumor therapeutic agent is not previously known.
This invention also provides an antitumor therapeutic agent suitable for combination therapy with a protein kinase C inhibitors capable of potentiating apoptosis in tumor cells determined by the above-described methods.
This invention also provides a pharmaceutical composition comprising an effective amount of the antitumor therapeutic agent determined by the above-described methods, a protein kinase C inhibitor capable of potentiating apoptosis in tumor cells and a pharmaceutically acceptable carrier.
This invention provides a method for enhancing therapy in a tumor bearing subject comprising administering to the subject an effective amount of a specific protein kinase C inhibitor capable of potentiating apoptosis in tumor cells during or prior to the treatment of an antitumor therapeutic agent. This invention provides the above method, wherein the specific protein kinase C inhibitor is Safingol (L-threo-dihydrosphingosine), R032-0432 (Bisindolylmaleimide tertiary amine), UCN-01 (7-OH-staurosporine), Flavopiridol (L86-8275), Bryostatin 1 (Macrocyclic lactone) or antisense nucleotides capable of inhibiting the expression of the protein kinase C.
In an embodiment, the antitumor therapeutic agent is a chemotherapeutic agent. In a further embodiment, the chemotherapeutic agent is selected from a group consisting of Mitomycin C, Carboplatin, Taxol and Doxorubicin. In a still further embodiment, the antitumor therapeutic agent is a radiotherapeutic agent
In an embodiment of the above-described methods, the tumor is a gastrointestinal cancer. In a further embodiment, the gastrointestinal cancer is gastric cancer, small bowel cancer, colon cancer or rectal cancer.
In a separate embodiment of the above-described methods, the tumor is a breast cancer. In another embodiment, the tumor is a ovarian cancer. In a still another embodiment, the tumor is of prostate cancer, lung cancer, melanoma, cervical carcinoma, pancreatic cancer, sarcoma, hepatoma, gallbladder cancer, leukemia or lymphoma.
Finally, this invention provides a method for potentiating apoptosis in tumor cells comprising contacting the cancerous cells with an effective amount of a specific protein kinase C inhibitor capable of potentiating apoptosis during or prior to the treatment of an antitumor therapeutic agent.